Sunday, 24 February 2013

Laboratory Techniques (Biochemistry)


BIOCHEMICAL TECHNIQUES:

These five tests identify the main biologically important chemical compounds. For each test take a small amount of the substance to test, and shake it in water in a test tube. If the sample is a piece of food, then grind it with some water in a pestle and mortar to break up the cells and release the cell contents. Many of these compounds are insoluble, but the tests work just as well on a fine suspension.
  • Starch (iodine test). To approximately 2 cm³ of test solution add two drops of iodine/potassium iodide solution. A blue-black colour indicates the presence of starch as a starch-polyiodide complex is formed. Starch is only slightly soluble in water, but the test works well in a suspension or as a solid.
  • Reducing Sugars (Benedict's test). All monosaccharides and most disaccharides (except sucrose) will reduce copper (II) sulphate, producing a precipitate of copper (I) oxide on heating, so they are called reducing sugars. Benedict’s reagent is an aqueous solution of copper (II) sulphate, sodium carbonate and sodium citrate. To approximately 2 cm³ of test solution add an equal quantity of Benedict’s reagent. Shake, and heat for a few minutes at 95°C in a water bath. A precipitate indicates reducing sugar. The colour and density of the precipitate gives an indication of the amount of reducing sugar present, so this test is semi-quantitative. The original pale blue colour means no reducing sugar, a green precipitate means relatively little sugar; a brown or red precipitate means progressively more sugar is present.
  • Non-reducing Sugars (Benedict's test). Sucrose is called a non-reducing sugarbecause it does not reduce copper sulphate, so there is no direct test for sucrose. However, if it is first hydrolysed (broken down) to its constituent monosaccharides (glucose and fructose), it will then give a positive Benedict's test. So sucrose is the only sugar that will give a negative Benedict's test before hydrolysis and a positive test afterwards. First test a sample for reducing sugars, to see if there are any present bef7ore hydrolysis. Then, using a separate sample, boil the test solution with dilute hydrochloric acid for a few minutes to hydrolyse the glycosidic bond. Neutralise the solution by gently adding small amounts of solid sodium hydrogen carbonate until it stops fizzing, then test as before for reducing sugars.
  • Lipids (emulsion test). Lipids do not dissolve in water, but do dissolve in ethanol. This characteristic is used in the emulsion test. Do not start by dissolving the sample in water, but instead shake some of the test sample with about 4 cm³ of ethanol. Decant the liquid into a test tube of water, leaving any undissolved substances behind. If there are lipids dissolved in the ethanol, they will precipitate in the water, forming a cloudy white emulsion.
  • Protein (biuret test). To about 2 cm³ of test solution add an equal volume of biuret solution, down the side of the test tube. A blue ring forms at the surface of the solution, which disappears on shaking, and the solution turns lilac-purple, indicating protein. The colour is due to a complex between nitrogen atoms in the peptide chain and Cu2+ ions, so this is really a test for peptide bonds.

Chromatography:

Chromatography is used to separate pure substances from a mixture of substances, such as a cell extract. It is based on different substances having different solubilities in different solvents. A simple and common form of chromatography uses filter paper.
  1. Pour some solvent into a chromatography tank and seal it, so the atmosphere is saturated with solvent vapour. Different solvents are suitable for different tasks, but they are usually mixtures of water with organic liquids such as ethanol or propanone.
  2. Place a drop of the mixture to be separated onto a sheet of chromatography paper near one end. This is the origin of the chromatogram. The spot should be small but concentrated. Repeat for any other mixtures. Label the spots with pencil, as ink may dissolve.
  3. Place the chromatography sheet into the tank so that the origin is just above the level of solvent, and leave for several hours. The solvent will rise up the paper by capillary action carrying the contents of the mixture with it. Any solutes dissolved in the solvent will be partitioned between the organic solvent (the moving phase) and the water, which is held by the paper (the stationary phase). The more soluble a solute is in the solvent the further up the paper it will move.
  4. When the solvent has nearly reached the top of the paper, the paper is removed and the position of the solvent front marked. The chromatogram may need to be developed to make the spots visible. For example amino acids stain purple with ninhydrin.
  5. The chromatogram can be analysed by measuring the distance travelled by the solvent front, and the distance from the origin to the centre of each spot. This is used to calculate the Rf (relative front) value for each spot:
An Rf value is characteristic of a particular solute in a particular solvent. It can be used to identify components of a mixture by comparing to tables of known Rf values.

Sometimes chromatography with a single solvent is not enough to separate all the constituents of a mixture. In this case the separation can be improved by two-dimensional chromatography, where the chromatography paper is turned through 90° and run a second time in a second solvent. Solutes that didn't separate in one solvent will separate in another because they have different solubilities.
There are many different types of chromatography.

  • Paper chromatography is the simplest, but does not always give very clean separation.

  • Thin layer chromatography (tlc) uses a thin layer of cellulose or silica coated onto a plastic or glass sheet. This is more expensive, but gives much better and more reliable separation.

  • Column chromatography uses a glass column filled with a cellulose slurry. Large samples can be pumped through the column and the separated fractions can be collected for further experiments, so this is preparative chromatography as opposed to analytical chromatography.
  • High performance liquid chromatography (HPLC) is an improved form of column chromatography that delivers excellent separation very quickly.

  • Electrophoresis uses an electric current to separate molecules on the basis of charge. It can also be used to separate on the basis of molecular size, and as such is used in DNA sequencing.

CELL FRACTIONATION:

This means separating different parts and organelles of a cell, so that they can be studied in detail. All the processes of cell metabolism (such as respiration or photosynthesis) have been studied in this way. The most common method of fractionating cells is to use differential centrifugation:

A more sophisticated separation can be performed by density gradient centrifugation. In this, the cell-free extract is centrifuged in a dense solution (such as sucrose or caesium chloride). The fractions don't pellet, but instead separate out into layers with the densest fractions near the bottom of the tube. The desired layer can then be pipetted off. This is the technique used in the Meselson-Stahl experiment (module 2) and it is also used to separate the two types of ribosomes. The terms 70S and 80S refer to their positions in a density gradient.

ENZYME KINETICS:
This means measuring the rate of enzyme reactions.

  • Firstly you need a signal to measure that shows the progress of the reaction. The signal should change with either substrate or product concentration, and it should preferably be something that can be measured continuously. Typical signals include colour changes, pH changes, mass changes, gas production, volume changes or turbidity changes. If the reaction has none of these properties, it can sometimes be linked to a second reaction which does generate one of these changes.
  • If you mix your substrate with enzyme and measure your signal, you will obtain a time-course. If the signal is proportional to substrate concentration it will start high and decrease, while if the signal is proportional to product it will start low and increase. In both cases the time-course will be curved (actually an exponential curve).
·         How do you obtain a rate from this time-course? One thing that is not a good idea is to measure the time taken for the reaction, for as the time-course shows it is very difficult to say when the reaction ends: it just gradually approaches the end-point. A better method is to measure theinitial rate - that is the initial slope of the time-course. This also means you don't need to record the whole time-course, but simply take one measurement a short time after mixing.
  • Repeat this initial rate measurement under different conditions (such as different substrate concentrations) and then plot a graph of rate vs. the factor. Each point on this second graph is taken from a separate initial rate measurement (or better still is an average of several initial rate measurements under the same conditions). Draw a smooth curve through the points.
Be careful not to confuse the two kinds of graph (the time-course and rate graphs) when interpreting your data.
One useful trick is to dissolve the substrate in agar in an agar plate. If a source of enzyme is placed in the agar plate, the enzyme will diffuse out through the agar, turning the substrate into product as it goes. There must be a way to distinguish the substrate from the product, and the reaction will then show up as a ring around the enzyme source. The higher the concentration of enzyme, the higher the diffusion gradient, so the faster the enzyme diffuses through the agar, so the larger the ring in a given time. The diameter of the ring is therefore proportional to the enzyme concentration. This can be done for many enzymes, e.g. a protein agar plate can be used for a protease enzyme, or a starch agar plate can be used for the enzyme amylase.

MICROSCOPY

Of all the techniques used in biology microscopy is probably the most important. The vast majority of living organisms are too small to be seen in any detail with the human eye, and cells and their organelles can only be seen with the aid of a microscope. Cells were first seen in 1665 by Robert Hooke (who named them after monks' cells in a monastery), and were studied in more detail by Leeuwehoek using a primitive microscope.

Units of measurement. The standard SI units of measurement used in microscopy are:

metre
m
= 1 m
millimetre
mm
= 10-3 m
micrometre
mm
= 10-6 m
nanometre
nm
= 10-9 m
picometre
pm
= 10-12 m
angstrom
Å
= 10-10 m (obsolete)

Magnification and Resolving Power. By using more lenses microscopes can magnify by a larger amount, but this doesn't always mean that more detail can be seen. The amount of detail depends on the resolving power of a microscope, which is the smallest separation at which two separate objects can be distinguished (or resolved). It is calculated by the formula:
where l is the wavelength of light, and n.a. is the numerical aperture of the lens (which ranges from about 0.5 to 1.4). So the resolving power of a microscope is ultimately limited by the wavelength of light (400-600nm for visible light). To improve the resolving power a shorter wavelength of light is needed, and sometimes microscopes have blue filters for this purpose (because blue has the shortest wavelength of visible light).

Different kinds of Microscope.

Light Microscope. This is the oldest, simplest and most widely-used form of microscopy. Specimens are illuminated with light, which is focussed using glass lenses and viewed using the eye or photographic film. Specimens can be living or dead, but often need to be stained with a coloured dye to make them visible. Many different stains are available that stain specific parts of the cell such as DNA, lipids, cytoskeleton, etc. All light microscopes today are compound microscopes, which means they use several lenses to obtain high magnification. Light microscopy has a resolution of about 200 nm, which is good enough to see cells, but not the details of cell organelles. There has been a recent resurgence in the use of light microscopy, partly due to technical improvements, which have dramatically improved the resolution far beyond the theoretical limit. For example fluorescence microscopy has a resolution of about 10 nm, while interference microscopy has a resolution of about 1 nm.

Electron Microscope. This uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen. This may seem strange, but electrons behave like waves and can easily be produced (using a hot wire), focussed (using electromagnets) and detected (using a phosphor screen or photographic film). A beam of electrons has an effective wavelength of less than 1 nm, so can be used to resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s revolutionised biology, allowing organelles such as mitochondria, ER and membranes to be seen in detail for the first time.
The main problem with the electron microscope is that specimens must be fixed in plastic and viewed in a vacuum, and must therefore be dead. Other problems are that the specimens can be damaged by the electron beam and they must be stained with an electron-dense chemical (usually heavy metals like osmium, lead or gold). Initially there was a problem of artefacts (i.e. observed structures that were due to the preparation process and were not real), but improvements in technique have eliminated most of these.
There are two kinds of electron microscope. The transmission electron microscope (TEM) works much like a light microscope, transmitting a beam of electrons through a thin specimen and then focussing the electrons to form an image on a screen or on film. This is the most common form of electron microscope and has the best resolution. The scanning electron microscope (SEM) scans a fine beam of electron onto a specimen and collects the electrons scattered by the surface. This has poorer resolution, but gives excellent 3-dimentional images of surfaces.
  • X-ray Microscope. This is an obvious improvement to the light microscope, since x-rays have wavelengths a thousand time shorter than visible light, and so could even be used to resolve atoms. Unfortunately there are no good x-ray lenses, so an image cannot be focussed, and useable x-ray microscopes do not yet exist. However, x-rays can be used without focussing to give a diffraction pattern, which can be used to work out the structures of molecules, such as those of proteins and DNA. 
  • Scanning Tunnelling Microscope (or Atomic Force Microscope). This uses a very fine needle to scan the surface of a specimen. It has a resolution of about 10 pm, and has been used to observe individual atoms for the first time.

Comparison of Light and Electron Microscopes


LIGHT MICROSCOPE
ELECTRON MICROSCOPE
illumination and source
light from lamp
electrons from hot wire
focusing
glass lenses
electromagnets
detection
eye or film
phosphor screen or film
magnification
1 500 x
500 000 x
resolution
200 nm
1 nm
specimen
living or dead
dead
staining
coloured dyes
heavy metals
cost
cheap to expensive
very expensive


Thursday, 21 February 2013

Cell Biology


Multiple choice Cells questions
1. To enter or leave a cell, substances must pass through
  • a. a microtubule.
  • b. the Golgi apparatus.
  • c. a ribosome.
  • d. the nucleus.
  • e. the plasma membrane.
2. Bacterial cell are prokaryotic; in comparison to a typical eukaryotic cell they would
  • a. be smaller.
  • b. have a smaller nucleus.
  • c. lack a plasma membrane.
  • d. have fewer internal membranous compartments.
  • e. have a greater variety of organelles.
3. You would expect a cell with an extensive Golgi apparatus to
  • a. make a lot of ATP.
  • b. secrete a lot of material.
  • c. move actively.
  • d. perform photosynthesis.
  • e. store large quantities of food
4. Which of the following correctly matches an organelle with its function?
  • a. mitochondrion . . . photosynthesis
  • b. nucleus . . . cellular respiration
  • c. ribosome . . . manufacture of lipids
  • d. lysosome . . . movement
  • e. central vacuole . . . storage
5. Of the following organelles, which group is involved in manufacturing substances needed by the cell?
  • a. lysosome, vacuole, ribosome
  • b. ribosome, rough ER, smooth ER
  • c. vacuole, rough ER, smooth ER
  • d. smooth ER, ribosome, vacuole
  • e. rough ER, lysosome, vacuole
6. A cell has mitochondria, ribosomes, smooth and rough ER, and other parts. Based on this information, it could not be
  • a. a cell from a pine tree.
  • b. a grasshopper cell.
  • c. a yeast (fungus) cell.
  • d. a bacterium.
  • e. Actually, it could be any of the above.
7. The electron microscope has been particularly useful in studying bacteria, because
  • a. electrons can penetrate tough bacterial cell walls.
  • b. bacteria are so small.
  • c. bacteria move so quickly they are hard to photograph.
  • d. with few organelles present, bacteria are distinguished by differences in individual macromolecules.
  • e. their organelles are small and tightly packed together
8. Cell fractionation is the most appropriate procedure for preparing ____ for study.
  • a. isolated cells which are normally found tightly attached to neighbouring cells
  • b. cells without a functional cytoskeleton
  • c. isolated organelles
  • d. the basic macromolecules
  • e. bone and other similar cells which are situated within a mineral framework
9. Which of the following clues would tell you whether a cell is prokaryotic or eukaryotic?
  • a. the presence or absence of a rigid cell wall
  • b. whether or not the cell is partitioned by internal membranes
  • c. the presence or absence of ribosomes
  • d. whether or not the cell carries out cellular metabolism
  • e. whether or not the cell contains DNA
10. Sara would like to film the movement of chromosomes during cell division. Her best choice for a microscope would be a
  • a. light microscope, because of its resolving power.
  • b. transmission electron microscope, because of its magnifying power.
  • c. scanning electron microscope, because the specimen is alive.
  • d. transmission electron microscope, because of its great resolving power.
  • e. light microscope, because the specimen is alive.

Chemistry of life: Terms(Questions) & Definitions


Terms
Definitions

What are the 4 most commonly occurring elements in living things?
Carbon, hydrogen, oxygen, nitrogen.

What are 5 other elements living things require?
Sulfur, calcium, phosphorus, iron, sodium.

What is the function of sulfur?
Used in proteins in prokaryote, animal and plant cells.

What is the function of calcium?
Flagella movement in prokaryotes forms cell plate during cytokinesis in plants, used in shells, bones, teeth and vesicle fusion in animals.

What is the function of phosphorus?
Nucleic acids and ATP in animals, plants and prokaryotes.

What is the function of iron?
Cytochrome (used in respiration) in palnts, and in cytochrome for mitochondrial respiration in plants and animals, hemoglobin.

What is the function of sodium?
Main cation in cytoplasm of plant cells, nerve impulse transmission in animals.

Why is water polar?
Because it has positive and negative ends/poles.

Why is water cohesive?
Because the negative end of one molecule's oxygen can form a hydrogen bond with the positive hydrogen of another molecule.

What can be said about water's specific heat capacity?
It is high i.e. it can store a lot of heat.

What solvent property is water given by its polarity?
It can dissolve other polar molecules and ions such as sugars, amino acids and sodium ions.

Give the three main functions of water in organisms.
Coolant, medium for metabolic reactions and a transport medium.

Why can water be used as a coolant?
It requires a high input of energy to break the hydrogen bonds and turn it into a vapour; so, evaporation of water off of the surface of an organism allows it to lose heat.

Give an example of water being used as a coolant.
Plants in deserts increase transpiration when in danger of overheating.

Why can water be used as a medium for metabolic reactions?
It is a good solvent, and is a liquid between 0-100 degrees centigrade, the temperature in most regions of the Earth.

What function does a watery habitat serve for organisms?
It dissolves substances which can then be absorbed by organisms.

What function does a watery cytoplasm serve for organisms?
It dissolves substances, and metabolic reactions can take place easily between substances dissolved in a liquid medium.

Why can water be a transport medium?
Its high specific heat capacity means it can store heat energy, and so organisms use if for heat transport (e.g. in blood). It is a good solvent and a dense medium, so it can dissolve substances and support heavy particles, and the cohesive property of water creates the transpiration stream in plants.

What are organic compounds?
Those that contain carbon and are found in living organisms.

What do organic compounds not contain?
Carbonates, hydrogencarbonates and oxides of carbon.

What do amino acids contain?
Nitrogen and a variable R side group.

How many amino acids are there which are used in proteins?
20.

What is the formula of glucose?
C6H12O6

What is the formula of ribose?
C5H10O5

What functional groups do fatty acids have?
A CH3 on one side and a COOH on the other.

How many CH2 groups are there in fatty acids?
Variable, but usually between 12 and 22.

What other groups can fatty acids have?
CH groups with double bonds between adjacent carbons.

What is an unsaturated fatty acid?
One with double bonds.

What is a saturated fatty acid?
One without double bonds.

What is a polyunsaturated fatty acid?
One with a large number of double bonds.

What are the three main types of carbohydrate?
Monosaccharides, disaccharides, polysaccharides.

Name 3 monosaccharides.
Glucose, galactose, fructose.

Name 3 disaccharides.
Maltose (2xglucose), lactose (glucose+galactose), sucrose (glucose+fructose).

Name 3 polysaccharides.
Starch, glycogen, cellulose (all poly-glucose).

Give a function of glucose.
It is broken down in animal respiration to release energy.

Give a function of lactose.
It is a sugar in milk produced by mammals.

Give a function of glycogen.
It is an energy store in liver and skeletal muscles.

Give a function of fructose.
It is an energy source for plants and a component of sucrose.

Give a function of sucrose.
It is un reactive and so is transported around the plant.

Give a function of cellulose.
It is the main component of cell walls.

What happens during condensation?
2 units are joined together with the release of water.

What happens during hydrolysis?
2 units are separated using water.

What are 6 functions of lipids?
Cuticle on leaf to prevent water loss, thermal insulation in animals as sub-cutaneous fat, energy store in plants and animals, oil on feathers and fur for water-proofing, main component of myelin sheath of neurons, buoyancy in aquatic animals.

Give 4 features of using carbohydrates as energy stores.
17kJ/g energy released, easily built up and broken down, present as glycogen in animals and starch in plants, converted to glucose when energy is needed.

Give 4 features of using lipids as energy stores.
38kJ/g energy released, hence more efficient than carbohydrate, hydrophobic, so less mass taken up storing water, metabolic pathways for build up and breakdown more complex and slower, converted to fatty acids and glycerol when energy is needed, then to coenzyme A.

What are the three parts of a nucleotide?
A phosphate group, a deoxyribose sugar and a base.

What links the sugar to the base and phosphate?
Strong covalent bonds.

What are the four bases in DNA?
Adenine, thymine, cytosine, guanine.

How does the double helix form?
Complementary base pairing (A-T, C-G), where the 2 sugar phosphate chains are anti-parallel. Weak hydrogen bonds form between the bases to hold the chains together.

What are cytosine and thymine?
Pyramidines.

What are adenine and guanine?
purines.

How many hydrogen bonds does the A-T connection have?
2.

How many hydrogen bonds does the C-G connection have?
3.

What directions do the DNA strands have?
One is 5'-3', the other is 3'-5'.

What does 5' mean?
Carbon 5 of the sugar has a phosphate attached and nothing else - it is a 'free end'.

What does 3' mean?
Carbon 3 contains a hydroxyl group and is the other 'free end'.

What is linked to the 5 and 3 carbons?
The sugars.

What do nucleosomes consist of?
A group of histone proteins with the DNA wrapped around; the DNA is locked in place by a second type of histone.

What are the 2 functions of nucelosomes?
They help supercoil the chromosomes and help to regulate transcription.

What are the 2 types of nuclear DNA?
Unique or single-copy genes and highly repetitive sequences.

What does 55-95% of the DNA consist of?
Sequences, called genes that only have a single copy.

What do the single-copy genes code for?
The functional polypeptides used by the cell or body, such as structural proteins, transport proteins, enzymes, hormones.

What does 5-45% of the DNA consist of?
Highly repetitive sequences.

What are highly repetitive sequences?
They can be 5-300 base pairs long, and can be repeated a moderate number of times, or up to 10^5 times in a genome. The location of these sequences shows no apparent patter, and their function is generally unclear.

What can we use the highly repetitive sequences for?
DNA profiling.

What else do eukaryotic genes contain?
Introns and exons.

Is DNA replication conservative or semi-conservative, and what does this mean?
Semi-conservative; the DNA double helixes produced will both contain one strand of the old DNA and one new strand.

What is the first step of DNA replication?
DNA helicase unwinds the DNA double helix by breaking the bonds between the bases; this forms the replication fork.

what does DNA polymerase III do?
It adds on new nucleotides to create the complementary strand of DNA, with hydrogen bonds between the bases, i.e. adds deoxynucleoside triphosphates to the 3' end.

What is the genetic code?
The linear sequence of bases.

How is the genetic code preserved?
By complementary base pairing.

What direction does DNA replication occur in?
5'-3'.

What does RNA primase do?
Adds nucleoside triphosphates on the lagging strand to form an RNA primer.

What does DNA polymerase I do?
Removes the RNA primer, replaces it using deoxynucleoside triphosphates.

What does DNA ligase do?
Joins the Okazaki fragments together.

What are Okazaki fragments?
Short lengths of single-stranded DNA made on the lagging strand.

What are deoxynucleoside triphosphates?
The building blocks of DNA; consist of the deoxyribose sugar, with a base and three phosphates.

What happens when the deoxynucleoside triphosphates are attached to others in DNA synthesis?
The two phosphates are removed, leaving only one for the sugar-phosphate backbone.

What are nucleoside triphosphates?
The molecules used to synthesise RNA, which is the same thing with ribose sugar instead.

Does DNA replication occur at one point only?
No - it is initiated at many points within eukaryotic chromosomes.

What are the 3 structural differences between RNA and DNA?
RNA is single stranded (but can fold back on itself to form double-stranded regions), has the base uracil instead of thymine, and has the sugar ribose instead of deoxyribose.

What are the 3 main differences of transcription compared to DNA replication?
Only one strand of the DNA is copied, RNA nucleotides are used (there is a pool of these in the nucleoplasm), the enzyme RNA polymerase is used.

What are the 3 key steps of transcription?
DNA is unzipped by RNA polymerase, RNA polymerase builds mRNA by pairing mRNA nucleotides onto the strand of DNA opposite the desired gene, mRNA is released and leaves the nucleus.

How many RNA bases code for one amino acid?
3.

How many codes are there for stop codons?
3.

What are stop codons?
Markers of the end of the 'message' on mRNA.

How many codes are there in total?
64.

What is degeneracy?
When two codes code for the same amino acid.

Why is the code universal?
Because all organisms use this same code.

What is translation?
The process used to manufacture a polypeptide chain from the mRNA code.

What direction does transcription occur in?
5'-3'.

What is the sense strand?
The side of the DNA double helix that is a gene.

What is the antisense strand?
The complementary sequence of bases, which is transcribed into RNA.

Which strand has the same sequence of bases as the RNA strand?
The sense strand, replacing T with U.

What is the promoter region?
A specific sequence of DNA bases at the start of a gene on the sense strand where RNA polymerase binds.

What is the purpose of RNA polymerase?
Adds nucleoside triphosphates using base pairing to the DNA template. It can only bind to DNA in the presence of other special proteins made by genes elsewhere in the genome.

What does the RNA polymerase do as it moves forwards?
It unwinds and separates the DNA at the front, and rewinds it at the back.

When does the RNA separate from the DNA?
As it is synthesised.

What is the terminator region?
A specific sequence of DNA bases marking the end of the transcription process on the sense strand.

What happens to the RNA polymerase when it reaches the terminator region?
It breaks free and the mRNA is released.

What does primary RNA contain?
Introns.

What are introns?
Sequences that are not translated into part of the protein, and must be removed.

What do the exons do?
They make up the mature RNA.

Where does the post-transcriptional modification of mRNA take place?
In the nucleus.

How many types of tRNA are there?
61, as there are 61 codons (excluding stop codons).

Where does the amino acid join on the tRNA?
At the 3' end.

What does adding the amino acid require?
Energy from ATP and a specific enzyme.

What are ribosomes composed of?
Ribosomal RNA and protein, in two subunits.

What does the small subunit of the ribosome do?
Binds to the mRNA.

What does the large subunit of the ribosome do?
Has three binding sites to bind to tRNA.

Where are ribosomes manufactured?
In the nucleolus within the nucleus.

What are the 4 stages of transcription?
Initiation, elongation, translocation and termination.

What happens during initiation?
The small ribosome subunit binds to mRNA and the first charged tRNA binds to the start codon (charged means it has an amino acid attached).

What is the start codon in prokaryotes?
AUG.

What does initiation form?
The initiation complex.

Where does initiation take place?
In the cytoplasm, where the ribosomal subunits are.

What happens during elongation?
The large ribosome subunit attaches so that the 1st charged tRNA is in binding site 1 (in the middle). The second charged tRNA binds in the second binding site (on the right). A peptide bond is formed between the two amino acids.

What is a peptide bond?
A bond between the C of a C=O and the N of an N-H.

Where is the polypeptide synthesised if it is designed for expot?
on the rER.

How is it known that a polypeptide is for export?
The first part of the polypeptide is a signal that causes the ribosome to bind to the rER.

What happens to the polypeptide as it is released if it has been synthesised on the rER?
It is passed through a protein channel in the rER.

What happens during translocation?
The mRNA is moved along one codon, and so the ribosome has moved along one codon in the 5'-3' direction. The uncharged tRNA is now in the 3rd site and is separated from the mRNA. It breaks free and picks up another amino acid from the cytoplasm. The 3rd charged tRNA moves into the 2nd binding site, and this process repeats.

What happens during termination?
The ribosome reaches the stop codon. There are no tRNAs with an anticodon for a stop codon. Release factors bind to site 2, and the ribosome subunits break free and the polypeptide is released.

What kind of polypeptides do free ribosomes synthesise?
Those primarily for use within the cell and cytoplasm.

What kind of polypeptides do ribosomes bound to the rER synthesise?
Those primarily for secretion or lysosomes.

How does the cell overcome the need to produce many polypeptides in large quantities?
As it would be energetically inefficient for each mRNA to synthesis only one polypeptide before being destroyed, ribosomes join behind the first ribosome so that multiple copies of the polypeptide can be synthesised rapidly.

What are enzymes?
Large molecules folded to form a 3-dimensional globular structure that act as catalysts.

What is an enzyme's active site?
A specifically shaped "pocket" for that enzyme's substrate to fit into. The shape of the active site matches the shape of the substrate, so that substrates are brought together in the correct orientation.

What happens to the bonds in substrates when bound to an enzyme?
They are weakened, making a reaction easier.

Which 3 factors affect enzyme action?
Substrate concentration, pH, temperature.

How does substrate concentration affect enzyme action?
Amount of enzymes, and therefore active sites, is fixed. As substrate concentration increases, more collisions occur with the enzymes and so more reactions occur. At a point, all the active sites are occupied by substrate at any one time, and so there can be no further increases in reaction rate.

How does pH affect enzyme action?
The 3D shape of the enzyme is held in place by bonds, which are strongest at the optimum pH. Changing the pH affects these bonds and the shape of the active site; if the substrate can no longer bind to the active site, reaction rate drops.

How does temperature affect enzyme action?
Temperature increases the molecular movement, and so the molecules in the solution collide with more energy, so more reactions occur. However, beyond the optimum temperature, the amino acids in the protein are moving so much that weak bonds are broken and the molecule begins to fall apart. The shape of the active site no longer fits the substrate and the reaction cannot take place.

Define "denaturation"
The irreversible, structural change in an enzyme that makes it unable to catalyse due to the substrate no longer fitting its active site.

Give a practical use of enzymes.
Production of lactose-free milk.

Why is lactose-free milk needed?
Some adults are intolerant to lactose, as the gene producing lactase gets switched off with age, and as lactose is a disaccharide it cannot be absorbed in the gut. The lactose is then fermented by bacteria in the large intestine resulting in nausea, abdominal pain and diarrhea.

How can milk be made lactose-free?
It is treated with lactase; this breaks down lactose to the monosaccharides glucose and galactose, which are easily absorbed by the gut.

Where is the lactase obtained from?
The fungus Kluyveromyces lactis.

Practically, how is the milk treated with lactase?
The lactase is immobilized and the milk is passed over it; this prevents the lactase from being in the product and is more economical, as it can then be reused.

What are 3 commercial applications of lactose-free milk?
Making ice cream (lactose crystallises and makes the ice cream grainy, whereas glucose and galactose stag dissolved). Yoghurt production, as bacteria can ferment glucose and galactose quicker than lactose, making production quicker. Lactose is also less sweet-tasting than glucose and galactose, so using lactose-free milk means less added sugar has to be added to products.

What do metabolic pathways consist of?
Chains and cycles of enzyme-catalysed reactions.

What does the "induced-fit model" mean?
The shape of the active site only corresponds to the shape of the substrate once it binds, which prevents possible but undesirable substrates from binding. The active site is flexible, so it allows a group of related molecules with similar shapes to be able to bind (e.g. peptidases). The reduces the number of different types of enzymes needed.

What is needed for a reaction to occur?
The necessary activation energy.

What do enzymes do to the activation energy?
They lower it, so that the reaction can take place at lower physiological temperature (usually between 0 and 40 degrees celsius).

What are the two types of enzyme inhibition?
Competitive and non-competitive.

What happens in competitive inhibition?
The substrate and inhibitor have similar shapes and compete for the active site.

What is an example of competitive inhibition?
In the liver, the metabolic pathway is: ethanol -> acetaldehyde -> acetate. Aldehyde dehydrogenase is the enzyme to convert acetaldehyde to acetate. Disulfiram is a drug used to help recovering alcoholics; it blocks the active side of aldehyde dehydrogenase, so acetaldehyde is not broken down in the liver and its accumulation in the blood causes severe headaches and nausea.

What happens in non-competitive inhibition?
The inhibitor has its own binding site, and binding of the inhibitor causes a conformational change in the shape of the active site, preventing the binding of the substrate.

Give an example of non-competitive inhibition.
Enzyme: acetylcholinesterase, substrate: acetylcholine, inhibitor: nerve gas Sarin.

What is the difference in effect of non-competitive and competitive inhibition at high concentrations?
Non-competitive inhibition will always reduce the rate of reaction at any concentration as the inhibitor binds to a separate site, whereas at higher substrate concentrations, the concentration of a competitive inhibitor becomes so low that it has no effect.

Where is non-competitive inhibition often used in the body?
To regulate metabolic pathways.

What are enzymes that are inhibited non-competitively in metabolic pathways called?
Allosteric enzymes.

What is the inhibitor for allosteric enzymes called?
The effector.

How do allosteric enzymes work?
The enzyme has two subunits, one with the substrate active site and the other with the allosteric site, where the effector binds (there may be more than one of these). The enzyme can alternate between an active form, which reacts with the substrate, and an inactive form, which does not. The effector can be an activator or inhibitor of the enzyme: the activator stabalises the active form, and the inhibitor stabalises the inactive form.

Give an example of an allosteric enzyme.
Phosphorylase is an enzyme in muscle which removes a glucose phosphate from the end of glycogen at the start of glycolysis. If the muscle is resting, its relative concentration of ATP will be high. If it is active, it will use up ATP and its relative concentration of AMP (adenosine monophosphate) will be high. AMP is the activator and ATP is the inhibitor; the enzyme is thus regulated so that glycogen is not broken down unnecessarily.